Methods of using GPR101 receptors to identify modulators of hypothalamic proopiomelanocortin (POMC)-derived biologically active peptide secretion

Abstract

The present invention relates to methods of using GPR101 G protein-coupled receptor (GPCR) to screen candidate compounds as modulators of hypothalamic proopiomelanocortin (POMC)-derived biologically active peptide secretion. Modulators of GPR101 receptor modulate hypothalamic POMC-derived biologically active peptide secretion and are useful in the treatment of POMC-derived biologically active peptide-related disorders. POMC-derived biologically active peptides include, but are not limited to, α-melanocyte stimulating hormone (α-MSH), β-melanocyte stimulating hormone (β-MSH) and γ-melanocyte stimulating hormone (γ-MSH). Agonists and partial agonists of GPR101 receptor stimulate hypothalamic α-MSH, β-MSH and γ-MSH secretion and are useful, for example, in the treatment and prevention of obesity and conditions related thereto (including but not limited to Type 2 diabetes, insulin resistance, and metabolic syndrome), inflammation-associated disorders, and pyrexia. Inverse agonists and antagonists of GPR101 receptor inhibit α-MSH, β-MSH and γ-MSH secretion and are useful, for example, in the treatment and prevention of disorders such as cachexia.

1. A method of identifying a candidate compound that stimulates hypothalamic proopiomelanocortin (POMC)-derived biologically active peptide secretion, said method comprising:
 (a) contacting the candidate compound with a recombinant host cell or a membrane thereof that comprises a G protein-coupled receptor (GPCR), wherein said receptor comprises an amino acid sequence selected from the group consisting of:
 (i) the amino acid sequence of SEQ ID NO:2; 
 (ii) the amino acid sequence of a GPCR encoded by a polynucleotide that is amplifiable by polymerase chain reaction (PCR) on a human genomic DNA sample using primers having the nucleotide sequence set forth in SEQ ID NO: 7 and SEQ ID NO: 8; 
 (iii) the amino acid sequence of SEQ ID NO: 4; 
 (iv) the amino acid sequence of SEQ ID NO: 6; 
 (v) the amino acid sequence of a GPCR encoded by a polynucleotide hybridizing under stringent conditions to the full-length complement of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5; 
 (vi) the amino acid sequence of a GPCR that has an amino acid sequence having at least 70% identity to SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6; and 
 (vii) the amino acid sequence of SEQ ID NO:2, but wherein the alanine at amino acid position 398 of SEQ ID NO:2 is substituted with an amino acid selected from the group consisting of lysine, arginine, and histidine, 
 wherein the receptor couples to a G protein or, wherein the GPCR induces cAMP accumulation; and 
 
 (b) determining the ability of the candidate compound to stimulate functionality of the GPCR; 
 (c) administering a vertebrate with a candidate compound that stimulates functionality of the GPCR; and 
 (d) measuring an energy homeostasis-related parameter of the vertebrate, wherein the energy homeostasis-related parameter is selected from the group consisting of body mass, adiposity, and percentage body fat due to food intake, 
 wherein the ability of the candidate compound to decrease an energy homeostasis-related parameter of the vertebrate is indicative of the candidate compound being a compound that stimulates hypothalamic POMC-derived biologically active peptide secretion. 
 
     
     
       2. The method of  claim 1 , wherein the GPCR comprises the amino acid sequence of SEQ ID NO: 2. 
     
     
       3. The method of  claim 1 , wherein the GPCR comprises the amino acid sequence of a GPCR that has an amino acid sequence having at least 70% identity to SEQ ID NO: 2. 
     
     
       4. The method of  claim 3 , wherein the GPCR comprises an amino acid sequence having at least 85% identity to SEQ ID NO: 2. 
     
     
       5. The method of  claim 1 , wherein the POMC-derived biologically active peptide is selected from the group consisting of adrenocorticotropic hormone (ACTH), β-endorphin, alpha-melanocyte stimulating hormone (α-MSH), beta-melanocyte stimulating hormone (β-MSH) and gamma-melanocyte stimulating hormone (γ-MSH). 
     
     
       6. The method of  claim 1 , wherein said determining is through the measurement of the level of a second messenger. 
     
     
       7. The method of  claim 6 , wherein the second messenger is cAMP. 
     
     
       8. The method of  claim 1 , wherein said determining is by using a Melanophore assay or by measuring GTPγS binding to a membrane comprising the GPCR or by using a cAMP-responsive reporter assay. 
     
     
       9. The method of  claim 1 , wherein the vertebrate is a non-human mammal. 
     
     
       10. The method of  claim 1 , wherein the vertebrate is a human. 
     
     
       11. The method of  claim 1 , wherein the method comprises identifying an agonist of the GPCR. 
     
     
       12. The method of  claim 11 , wherein the agonist is an agonist of human GPR101. 
     
     
       13. The method of  claim 1 , wherein the method comprises identifying a partial agonist of the GPCR. 
     
     
       14. The method of  claim 13 , wherein the partial agonist is a partial agonist of human GPR101. 
     
     
       15. A method of screening candidate compounds as pharmaceutical agents for a POMC-derived biologically active peptide-related disorder, said method comprising:
 (a) contacting the candidate compound with a recombinant host cell or a membrane thereof that comprises a G protein-coupled receptor (GPCR), wherein said receptor comprises an amino acid sequence selected from the group consisting of:
 the amino acid sequence of SEQ ID NO:2; 
 the amino acid sequence of a GPCR encoded by a polynucleotide that is amplifiable by polymerase chain reaction (PCR) on a human genomic DNA sample using primers having the nucleotide sequence set forth in SEQ ID NO: 7 and SEQ ID NO: 8; 
 (iii) the amino acid sequence of SEQ ID NO: 4; 
 (iv) the amino acid sequence of SEQ ID NO: 6; 
 (v) the amino acid sequence of a GPCR encoded by a polynucleotide hybridizing under stringent conditions to the full-length complement of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5; 
 (vi) the amino acid sequence of a GPCR that has an amino acid sequence having at least 70% identity to SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6; and 
 (vii) the amino acid sequence of SEQ ID NO:2, but wherein the alanine at amino acid position 398 of SEQ ID NO:2 is substituted with an amino acid selected from the group consisting of lysine, arginine, and histidine, 
 wherein the receptor couples to a G protein or, wherein the GPCR induces cAMP accumulation; and 
 
 (b) detecting a ligand bound to said GPCR, 
 (c) administering a vertebrate with a ligand that binds to said GPCR; and 
 (d) measuring an energy homeostasis-related parameter of the vertebrate, wherein the energy homeostasis-related parameter is selected from the group consisting of body mass, adiposity, and percentage body fat due to food intake, 
 wherein the ability of the ligand to decrease an energy homeostasis-related parameter of the vertebrate is indicative of the ligand being suitable as a pharmaceutical agent for a POMC-derived biologically active peptide-related disorder. 
 
     
     
       16. The method of  claim 15 , wherein the GPCR comprises the amino acid sequence of SEQ ID NO: 2. 
     
     
       17. The method of  claim 15 , wherein the POMC-derived biologically active peptide is selected from the group consisting of adrenocorticotropic hormone (ACTH), β-endorphin, alpha-melanocyte stimulating hormone (α-MSH), beta-melanocyte stimulating hormone (β-MSH) and gamma-melanocyte stimulating hormone (γ-MSH). 
     
     
       18. The method of  claim 15 , wherein the GPCR comprises an amino acid sequence having at least 70% identity to SEQ ID NO: 2. 
     
     
       19. The method of  claim 18 , wherein the GPCR comprises an amino acid sequence having at least 85% identity to SEQ ID NO: 2. 
     
     
       20. The method of  claim 15 , wherein the ligand that decreases an energy homeostasis-related parameter of the vertebrate is a ligand of human GPR101. 
     
     
       21. The method of  claim 20 , wherein the ligand of human GPR101 is a selective ligand. 
     
     
       22. The method of  claim 15 , wherein the vertebrate is a non-human mammal. 
     
     
       23. The method of  claim 15 , wherein the vertebrate is a human. 
     
     
       24. A method of identifying a compound that stimulates hypothalamic POMC-derived biologically active peptide secretion, said method comprising:
 (a) measuring an energy homeostasis-related parameter of a vertebrate, the vertebrate having been administered a GPR101 ligand, wherein the energy homeostasis-related parameter is selected from the group consisting of body mass, adiposity, and percentage body fat due to food intake; 
 wherein the ability of the GPR101 ligand to decrease an energy homeostasis-related parameter of the vertebrate is indicative of a compound that stimulates hypothalamic POMC-derived biologically active peptide secretion. 
 
     
     
       25. The method of  claim 24 , wherein the vertebrate is a non-human mammal. 
     
     
       26. The method of  claim 24 , wherein the vertebrate is a human. 
     
     
       27. The method of  claim 24 , wherein the POMC-derived biologically active peptide is selected from the group consisting of adrenocorticotropic hormone (ACTH), β-endorphin, alpha-melanocyte stimulating hormone (α-MSH), beta-melanocyte stimulating hormone (β-MSH) and gamma-melanocyte stimulating hormone (γ-MSH). 
     
     
       28. The method of  claim 24 , wherein the GPR101 ligand that decreases an energy homeostasis-related parameter of the vertebrate is further identified as a ligand of human GPR101. 
     
     
       29. The method of  claim 24 , wherein the GPR101 ligand that decreases an energy homeostasis-related parameter of the vertebrate is an antibody. 
     
     
       30. The method of  claim 24 , wherein the GPR101 ligand that decreases an energy homeostasis-related parameter of the vertebrate is a selective GPR101 ligand having a selectivity for GPR101 over GPR161. 
     
     
       31. The method of  claim 24 , wherein the method identifies said GPR101 ligand as a GPR101 agonist. 
     
     
       32. The method of  claim 31 , wherein the agonist has an EC50 of less than 10 μM. 
     
     
       33. The method of  claim 31 , wherein the agonist has an EC50 of less than 1 μM. 
     
     
       34. The method of  claim 31 , wherein the agonist has an EC50 of less than 100 nM. 
     
     
       35. The method of  claim 31 , wherein the agonist has a selectivity for GPR101 over GPR161 of at least 10-fold. 
     
     
       36. The method of  claim 31 , wherein the agonist is a GPR101 partial agonist. 
     
     
       37. The method of  claim 31 , wherein the agonist is an agonist of human GPR101. 
     
     
       38. A method of identifying a compound that stimulates hypothalamic proopiomelanocortin (POMC)-derived biologically active peptide secretion, said method comprising:
 (a) administering a vertebrate a GPR101 ligand; and 
 (b) measuring an energy homeostasis-related parameter of the vertebrate, wherein the energy homeostasis-related parameter is selected from the group consisting of body mass, adiposity, and percentage body fat due to food intake, 
 wherein the ability of the GPR101 ligand to decrease an energy homeostasis-related parameter of the vertebrate is indicative of the GPR101 ligand being a compound that stimulates hypothalamic POMC-derived biologically active peptide secretion. 
 
     
     
       39. The method of  claim 38 , wherein the vertebrate is a non-human mammal. 
     
     
       40. The method of  claim 38 , wherein the vertebrate is a human. 
     
     
       41. The method of  claim 38 , wherein the POMC-derived biologically active peptide is selected from the group consisting of adrenocorticotropic hormone (ACTH), β-endorphin, alpha-melanocyte stimulating hormone (α-MSH), beta-melanocyte stimulating hormone (β-MSH) and gamma-melanocyte stimulating hormone (γ-MSH). 
     
     
       42. The method of  claim 38 , wherein the GPR101 ligand is further identified as a ligand of human GPR101. 
     
     
       43. The method of  claim 38 , wherein the GPR101 ligand is an antibody. 
     
     
       44. The method of  claim 38 , wherein the GPR101 ligand is a selective GPR101 ligand, said selective. GPR101 ligand having a selectivity for GPR101 over GPR161. 
     
     
       45. The method of  claim 38 , wherein the method identifies said GPR101 ligand as a GPR101 agonist. 
     
     
       46. The method of  claim 45 , wherein the agonist has an EC50 of less than 10 μM. 
     
     
       47. The method of  claim 45 , wherein the agonist has an EC50 of less than 1 μM. 
     
     
       48. The method of  claim 45 , wherein the agonist has an EC50 of less than 100 nM. 
     
     
       49. The method of  claim 45 , wherein the GPR101 agonist has a selectivity for GPR101 over GPR161 of at least 10-fold. 
     
     
       50. The method of  claim 45 , wherein the agonist is an agonist of human GPR101. 
     
     
       51. The method of  claim 45 , wherein the agonist is a GPR101 partial agonist. 
     
     
       52. A method of identifying a compound that stimulates hypothalamic POMC-derived biologically active peptide secretion, said method comprising:
 (a) measuring a modulation of energy homeostasis in a vertebrate, the vertebrate having been administered a GPR101 ligand, wherein the modulation is selected from the group consisting of modulation of obesity, modulation of satiety, and modulation of hyperphagia, 
 wherein the ability of the GPR101 ligand to oppose obesity, to promote satiety, or to oppose hyperphagia in the vertebrate is indicative of the GPR101 ligand being a compound that stimulates hypothalamic POMC-derived biologically active peptide secretion. 
 
     
     
       53. The method of  claim 52 , wherein the vertebrate is a non-human mammal. 
     
     
       54. The method of  claim 52 , wherein the vertebrate is a human. 
     
     
       55. A method of identifying a compound that stimulates hypothalamic proopiomelanocortin (POMC)-derived biologically active peptide secretion, said method comprising:
 (a) administering a vertebrate a GPR101 ligand; and 
 (b) measuring a modulation of energy homeostasis in the vertebrate, wherein the modulation is selected from the group consisting of modulation of obesity, modulation of satiety, and modulation of hyperphagia, 
 wherein the ability of the GPR101 ligand to oppose obesity, to promote satiety, or to oppose hyperphagia in the vertebrate is indicative of the GPR101 ligand being a compound that stimulates hypothalamic POMC-derived biologically active peptide secretion. 
 
     
     
       56. The method of  claim 55 , wherein the vertebrate is a non-human mammal. 
     
     
       57. The method of  claim 55 , wherein the vertebrate is a human.